Non-phosphorylated Β-catenin Research Articles

EZH2 inhibition or genetic ablation suppresses cyst growth in autosomal dominant polycystic kidney disease

BackgroundAutosomal Dominant Polycystic Kidney Disease (ADPKD) is a prevalent genetic disorder characterized by the formation of renal cysts leading to kidney failure. Despite known genetic underpinnings, the variability in disease progression suggests additional regulatory layers, including epigenetic modifications.MethodsWe utilized various ADPKD models, including Pkd1 and Ezh2 conditional knockout (Pkd1delta/delta:Ezh2delta/delta) mice, to explore the role of Enhancer of Zeste Homolog 2 (EZH2) in cystogenesis. Pharmacological inhibition of EZH2 was performed using GSK126 or EPZ-6438 across multiple models.ResultsEZH2 expression was significantly upregulated in Pkd1−/− cells, Pkd1delta/delta mice, and human ADPKD kidneys. EZH2 inhibition attenuates cyst development in MDCK cells and a mouse embryonic kidney cyst model. Both Ezh2 conditional knockout and GSK126 treatment suppressed renal cyst growth and protected renal function in Pkd1delta/delta mice. Mechanistically, cAMP/PKA/CREB pathway increased EZH2 expression. EZH2 mediated cystogenesis by enhancing methylation and activation of STAT3, promoting cell cycle through p21 suppression, and stimulating non-phosphorylated β-catenin in Wnt signaling pathway. Additionally, EZH2 enhanced ferroptosis by inhibiting SLC7A11 and GPX4 in ADPKD.ConclusionOur findings elucidate the pivotal role of EZH2 in promoting renal cyst growth through epigenetic mechanisms and suggest that EZH2 inhibition or ablation may serve as a novel therapeutic approach for managing ADPKD.

Sclerostin antibody improves alveolar bone quality in the Hyp mouse model of X-linked hypophosphatemia (XLH)

X-linked hypophosphatemia (XLH) is a rare disease of elevated fibroblast growth factor 23 (FGF23) production that leads to hypophosphatemia and impaired mineralization of bone and teeth. The clinical manifestations of XLH include a high prevalence of dental abscesses and periodontal disease, likely driven by poorly formed structures of the dentoalveolar complex, including the alveolar bone, cementum, dentin, and periodontal ligament. Our previous studies have demonstrated that sclerostin antibody (Scl-Ab) treatment improves phosphate homeostasis, and increases long bone mass, strength, and mineralization in the Hyp mouse model of XLH. In the current study, we investigated whether Scl-Ab impacts the dentoalveolar structures of Hyp mice. Male and female wild-type and Hyp littermates were injected with 25 mg·kg−1 of vehicle or Scl-Ab twice weekly beginning at 12 weeks of age and euthanized at 20 weeks of age. Scl-Ab increased alveolar bone mass in both male and female mice and alveolar tissue mineral density in the male mice. The positive effects of Scl-Ab were consistent with an increase in the fraction of active (nonphosphorylated) β-catenin, dentin matrix protein 1 (DMP1) and osteopontin stained alveolar osteocytes. Scl-Ab had no effect on the mass and mineralization of dentin, enamel, acellular or cellular cementum. There was a nonsignificant trend toward increased periodontal ligament (PDL) attachment fraction within the Hyp mice. Additional PDL fiber structural parameters were not affected by Scl-Ab. The current study demonstrates that Scl-Ab can improve alveolar bone in adult Hyp mice.

C1orf109 promotes malignant phenotype of liver cancer via wnt signaling pathway in a CK2-dependent manner

Chromosome 1 open reading frame 109 (C1orf09) is a protein whose expression pattern and biological function in humans, particularly in malignant tumors, have not been explored. In this study, both bioinformatics and immunohistochemical staining revealed that C1orf109 was overexpressed in the cytoplasm of liver cancer cells, and the positive ratio of C1orf109 in liver cancer samples (42.5%, 37/87) was significantly higher than that in normal liver tissues (10%, 3/30, P = 0.0012). C1orf109 expression was correlated with an advanced TNM stage (P = 0.017) and vascular invasion (P = 0.023) and predicted the poor overall survival of patients with liver cancer (P = 0.001). C1orf109 facilitated tumor growth, colony formation, migration, and invasion by activating Wnt signaling by upregulating non-phosphorylated β-catenin and its downstream target genes such as CyclinD1, c-myc, and MMP7. Our results also suggest that C1orf109 interacts and co-localizes with casein kinase II (CK2) to activate Wnt signaling. Treatment with a CK2-specific inhibitor markedly counteracted the increased expression of CyclinD1, c-Myc, and MMP7, as well as the upregulation of tumor proliferation and invasion caused by C1orf109 overexpression. Taken together, our results indicate that C1orf109 accelerates liver cancer cell proliferation and invasion by strengthening the Wnt signaling pathway in a CK2-dependent manner.

Transient and Prolonged Activation of Wnt Signaling Contribute Oppositely to the Pathogenesis of Asherman's Syndrome.

Asherman’s Syndrome (AS) is caused by dysfunction of endometrial regenerative ability, which is controlled by adult stem cells and their niche. The Wnt signaling pathway has been demonstrated to be implicated in this process. This study aimed to clarify the relationship between the Wnt signaling pathway and the progression of AS after initial endometrial damage. Endometria with and without adhesion as well as from the intrauterine devices three months after the surgery were collected to compare the area of fibrosis. The area% of fibrosis did not vary significantly. Significantly higher expression of non-phosphorylated β-catenin, Wnt5a and Wnt7a was identified in the endometria with adhesion. The CD140b+CD146+ endometrial stem-like cells were present in the endometria with adhesion. Both Wnt5a and Wnt7a promoted stem cell proliferation. However, only Wnt7a preserved stem cell population by stimulating self-renewal. A rat endometrial injury model was established to investigate the effect of the activated Wnt/β-catenin signaling pathway on endometrial healing. We found that a transient activation of the Wnt/β-catenin signaling pathway promoted angiogenesis and increased the number of glands. In conclusion, transient activation of the Wnt/β-catenin signaling pathway during the acute endometrial damage may help the tissue regeneration, while prolonged activation may correlate to fibrosis formation.

SOX transcription factors direct TCF-independent WNT/β-catenin responsive transcription to govern cell fate in human pluripotent stem cells.

WNT/β-catenin signaling controls gene expression across biological contexts from development and stem cell homeostasis to diseases including cancer. How β-catenin is recruited to distinct enhancers to activate context-specific transcription is unclear, given that most WNT/ß-catenin-responsive transcription is thought to be mediated by TCF/LEF transcription factors (TFs). With time-resolved multi-omic analyses, we show that SOX TFs can direct lineage-specific WNT-responsive transcription during the differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm and neuromesodermal progenitors. We demonstrate that SOX17 and SOX2 are required to recruit β-catenin to lineage-specific WNT-responsive enhancers, many of which are not occupied by TCFs. At TCF-independent enhancers, SOX TFs establish a permissive chromatin landscape and recruit a WNT-enhanceosome complex to activate SOX/ß-catenin-dependent transcription. Given that SOX TFs and the WNT pathway are critical for specification of most cell types, these results have broad mechanistic implications for the specificity of WNT responses across developmental and disease contexts.

Pigment epithelium-derived factor modulates periodontal homeostasis in mice and induces osteogenic differentiation of human periodontal ligament fibroblasts

ABSTRACT Aim The aim of this study was to investigate the influence of pigment epithelium-derived factor (PEDF) on periodontal homeostasis in mice and the osteogenic differentiation of human periodontal ligament fibroblasts (PDLFs). Materials and Methods Micro-computed tomography and histology were performed to compare the alveolar bone volume, density, and bone-related markers between PEDF-deficient (PEDF−/−) and wild-type (WT) mice. Furthermore, after recombinant human PEDF treatment, the PDLF viability and osteogenic differentiation were examined using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, alkaline phosphatase (ALP) activity assay, Von Kossa staining, Alizarin red staining, real-time quantitative polymerase chain reaction (qRT-PCR), and immunoblotting. Results The alveolar bone volume and density of PEDF−/− mice were significantly lower than those of the WT mice. Higher receptor activator for nuclear factor-κB ligand (RANKL) expression and lower osteoprotegerin (OPG) expression levels were observed in the PEDF−/− group. Moreover, PEDF treatment did not affect the PDLF proliferation. PEDF dose-dependently improved mineral deposition. Compared with the control group, 250 ng/mL PEDF promoted OPG mRNA expression in PDLFs on Day 3 but inhibited RANKL, Wnt5a, GSK3b mRNA, and non-phosphorylated β-catenin protein expression. However, 250 ng/mL PEDF had no significant effect on the expression of Wnt3a. On Day 7, after culture with 250 ng/mL PEDF in osteogenic medium, the ALP and RUNX2 protein levels were upregulated. VEGF protein expression was reduced in a dose-dependent manner after PEDF stimulation. The PEDF protein expression increased as the osteogenic induction time increased. Conclusion PEDF gene knockout suppresses periodontal homeostasis in mice, and PEDF treatment induces PDLF osteogenic differentiation in vitro.

Heterozygous calcyclin-binding protein/Siah1-interacting protein (CACYBP/SIP) gene pathogenic variant linked to a dominant family with paucity of interlobular bile duct.

Paucity of interlobular bile ducts (PILBD) is a heterogeneous disorder classified into two categories, syndromic and non-syndromic bile duct paucity. Syndromic PILBD is characterized by the presence of clinical manifestations of Alagille syndrome. Non-syndromic PILBD is caused by multiple diseases, such as metabolic and genetic disorders, infectious diseases, and inflammatory and immune disorders. We evaluated a family with a dominantly inherited PILBD, who presented with cholestasis at 1-2 months of age but spontaneously improved by 1 year of age. Next-generation sequencing analysis revealed a heterozygous CACYBP/SIP p.E177Q pathogenic variant. Calcyclin-binding protein and Siah1 interacting protein (CACYBP/SIP) form a ubiquitin ligase complex and induce proteasomal degradation of non-phosphorylated β-catenin. Immunohistochemical analysis revealed a slight decrease in CACYBP and β-catenin levels in the liver of patients in early infancy, which almost normalized by 13 months of age. The CACYBP/SIP p.E177Q pathogenic variant may form a more active or stable ubiquitin ligase complex that enhances the degradation of β-catenin and delays the maturation of intrahepatic bile ducts. Our findings indicate that accurate regulation of the β-catenin concentration is essential for the development of intrahepatic bile ducts and CACYBP/SIP pathogenic variant is a novel cause of PILDB.

Excess folic acid supplementation before and during pregnancy and lactation activates β-catenin in the brain of male mouse offspring

Folic acid (FA) supplementation in early pregnancy is recommended to protect against birth defects. But excess FA has exhibited neurodevelopmental toxicity. We previously reported that the mice treated with 2.5-fold the dietary requirement of FA one week before mating and throughout pregnancy and lactation displayed abnormal behaviors in the offspring. Here we found the levels of non-phosphorylated β-catenin (active) were increased in the brains of weaning and adult FA-exposed offspring. Meanwhile, demethylation of protein phosphatase 2 A catalytic subunit (PP2Ac), which suppresses its enzyme activity in regulatory subunit dependent manner, was significantly inhibited. Among the upstream regulators of β-catenin, PI3K/Akt/GSK-3β but not Wnt signaling was stimulated in FA-exposed brains only at weaning. In mouse neuroblastoma N2a cells, knockdown of PP2Ac or leucine carboxyl methyltransferase-1 (LCMT-1), or overexpression of PP2Ac methylation-deficient mutant decreased β-catenin dephosphorylation. These results suggest that excess FA may activate β-catenin via suppressing PP2Ac demethylation, providing a novel mechanism for the influence of FA on neurodevelopment.

Biallelic ANKS6 mutations cause late-onset ciliopathy with chronic kidney disease through YAP dysregulation.

Nephronophthisis-related ciliopathies (NPHP-RC) comprises a group of inherited kidney diseases, caused by mutations in genes encoding proteins localizing to primary cilia. NPHP-RC represents one of the most frequent monogenic causes of renal failure within the first three decades of life, but its molecular disease mechanisms remain unclear. Here, we identified biallelic ANKS6 mutations in two affected siblings with late-onset chronic kidney disease by whole-exome sequencing. We employed patient-derived fibroblasts generating an in vitro model to study the precise biological impact of distinct human ANKS6 mutations, completed by immunohistochemistry studies on renal biopsy samples. Functional studies using patient-derived cells showed an impaired integrity of the ciliary inversin compartment with reduced cilia length. Further analyses demonstrated that ANKS6 deficiency leads to a dysregulation of Hippo-signaling through nuclear yes-associated protein (YAP) imbalance and disrupted ciliary localization of YAP. In addition, an altered transcriptional activity of canonical Wnt target genes and altered expression of non-phosphorylated (active) β-catenin and phosphorylated glycogen synthase kinase 3β were observed. Upon ciliation, ANKS6 deficiency revealed a deranged subcellular localization and expression of components of the endocytic recycling compartment. Our results demonstrate that ANKS6 plays a key role in regulating the Hippo pathway, and ANKS6 deficiency is linked to dysregulation of signaling pathways. Our study provides molecular clues in understanding pathophysiological mechanisms of NPHP-RC and may offer new therapeutic targets.

Simvastatin Inhibits Wnt/β-Catenin Pathway in Uterine Leiomyoma.

The Wnt/β-catenin pathway is upregulated in uterine leiomyomas, the most common benign tumors in the female reproductive tract. Simvastatin is an antihyperlipidemic drug, and previous in vitro and in vivo reports showed that it may have therapeutic effects in treating leiomyomas. The objective of this study was to examine the effects of simvastatin on the Wnt/β-catenin signaling pathway in leiomyoma. We treated primary and immortalized human leiomyoma cells with simvastatin and examined its effects using quantitative real-time polymerase chain reaction, Western blotting, and immunocytochemistry. We also examined the effects using human leiomyoma tissues from an ongoing randomized controlled trial in which women with symptomatic leiomyoma received simvastatin (40 mg) or placebo for 3 months prior to their surgery. The results of this study revealed that simvastatin significantly reduced the expression of Wnt4 and its co-receptor LRP5. After simvastatin treatment, levels of total β-catenin and its active form, nonphosphorylated β-catenin, were reduced in both cell types. Additionally, simvastatin reduced the expression of Wnt4 and total β-catenin, as well as nonphosphorylated β-catenin protein expression in response to estrogen and progesterone. Simvastatin also inhibited the expression of c-Myc, a downstream target of the Wnt/β-catenin pathway. The effect of simvastatin on nonphosphorylated-β-catenin, the key regulator of the Wnt/β-catenin pathway, was recapitulated in human leiomyoma tissue. These results suggest that simvastatin may have a beneficial effect on uterine leiomyoma through suppressing the overactive Wnt/β-catenin pathway.

TGF-Β SIGNALING REGULATES WNT/Β-CATENIN PATHWAY IN UTERINE LEIOMYOMA CELLS

Uterine leiomyomas, also known as uterine fibroids, are the most common benign gynecologic tumor of the female reproductive tract. Despite their high prevalence and heavy burden, their pathogenesis is not clear. Several signaling aberrations have been identified, including Wnt/β-catenin and TGF-β. TGF-β pathway is a crucial mediator of extracellular matrix (ECM) deposition and fibrosis and is upregulated in leiomyoma compared to the myometrium. The Wnt/β-catenin pathway is a conserved pathway implicated in proliferation, differentiation and stem cell renewal and has also been implicated in uterine leiomyoma development. Due to the importance of these two pathways in uterine leiomyoma development, we are interested in studying the interaction between these two pathways to better understand the pathogenesis of these tumors. After obtaining informed consent, primary fibroid and myometrial cells were isolated from hysterectomy surgeries at Johns Hopkins Hospital. Primary cells and immortalized fibroid (HuLM) and myometrial (UtSM) were treated with TGF-β3 (10ng/mL) for 48 hours. The effect of TGF-β3 on the Wnt/β-catenin pathway was examined using western blot through assessing the expression of total β-catenin, non-phosphorylated β-catenin, the active form of β-catenin, and Dickkopf-1 (DKK1), an inhibitor of Wnt pathway. The student’s t-test was used to determine statistically significant differences (P<0.05). As expected, TGF-β3 treatment induced the expression of ECM proteins collagen 1A and fibronectin in both primary and immortalized fibroid and myometrial cells. The expression of total β-catenin and non-phosphorylated β-catenin was higher in leiomyoma cells, and TGF-β3 treatment significantly increased the expression of total β-catenin and non-phosphorylated β-catenin in both myometrial by 66% and 59%, respectively and fibroid cells by 58% and 62%, respectively (p-value <0.05). The expression of the Wnt pathway inhibitor, DKK-1, was significantly lower in primary leiomyoma cells compared to myometrial cells, and TGF-β3 treatment resulted in a significant reduction in DKK-1 expression in both myometrial by 85% and fibroid cells by 61%(p-value <0.05). These results indicate that TGF-β3 and Wnt/β-catenin signal transduction pathways interact in uterine leiomyoma. Further analyses are needed to clarify the interaction and the role of DKK-1 in leiomyoma development.

Abstract 1110: Targeting β-catenin enhances the therapeutic efficacy of osimertinib in EGFR- mutant non-small cell lung cancer (NSCLC)

Abstract In the US, ~15% of patients with metastatic lung adenocarcinoma have tumors harboring “driver” mutations in the EGFR gene, which demonstrate major clinical responses to EGFR Tyrosine Kinase Inhibitors (TKIs). The third-generation EGFR TKI osimertinib shows superior clinical activity compared to early-generation EGFR TKIs, however, essentially all tumors eventually develop resistance to osimertinib. We previously demonstrated that EGFR TKIs induce “adaptive persistence” in cancer cells with stem-like properties, allowing these cells to survive and acquire resistance. Activation of β-catenin and downstream signaling components is one of the major pathways enabling this adaptive persistence. Our goal is to identify and validate β-catenin inhibitors that have the potential to improve the depth and duration of response to osimertinib in EGFR-mutant NSCLC. We used the EGFR-mutant NSCLC cell lines HCC827, HCC4006, and H1975 to evaluate the effects of treatment with osimertinib and a β-catenin inhibitor, BC2059. Cell viability assays were done to determine the IC50 values of BC2059 in this panel of cell lines. ALDEFLUOR assays were performed to assay for induced stem-ness. We also assessed β-catenin protein levels and downstream signaling events in various treatment conditions. To evaluate the preclinical activity of β-catenin inhibition, we treated mice bearing HCC4006 and HCC827 xenografts with osimertinib alone or in combination with BC2059. Preclinical activity was assessed through progression free survival and overall survival. Our in vitro studies demonstrated that NSCLC cell lines are sensitive to β-catenin inhibition in a time and dose-dependent manner, and the addition of BC2059 to osimertinib enhances tumor cell killing. Osimertinib treatment increased non-phosphorylated and total β-catenin levels in EGFR-mutant cell lines. We further showed that osimertinib treatment induced ALDH activity that was sensitive to β-catenin inhibition in HCC4006 and HCC827 cells. BC2059 decreased osimertinib-induced ALDH positivity by &amp;gt;10 fold in HCC4006 and ~2 fold in HCC827 cells. Osimertinib treatment increased levels of the secreted β-catenin transcriptional target PAI1 in cultured media, which was inhibited by BC2059. Our preclinical studies also demonstrated that the combination of osimertinib and BC2059 decreased tumor recurrence and improved overall survival in mice bearing EGFR-mutant human lung cancer xenografts. Overall, our studies show that osimertinib in combination with the β-catenin inhibitor BC2059 enhances osimertinib efficacy and delays tumor recurrence, possibly by targeting drug-tolerant persistent clones that are enriched for with EGFR TKI treatment alone. These data provide strong rationale for a clinical trial that evaluates the combination of osimertinib and BC2059 in patients with metastatic EGFR-mutant NSCLC. Citation Format: Shankar Suman, Shu-Xiao Guan, Nastaran Navari, Rajeswara Rao Arasada, David P. Carbone, Regan M. Memmott. Targeting β-catenin enhances the therapeutic efficacy of osimertinib in EGFR- mutant non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1110.

Novel Stem Cell-Targeted Therapy for Multiple Myeloma Based on Dual Inhibition of EZH1/2

Multiple myeloma (MM) is largely incurable because relapse eventually occurs, despite the recent development of novel therapies. This is mainly caused by a remaining population of drug-resistant myeloma stem cells. There is a side population (SP) enriched with myeloma stem cells. Therefore, targeting SP cells may be a promising strategy to prevent and treat MM relapse.Polycomb repressive complexes 1 (PRC1) and 2 (PRC2) are important epigenetic regulators that maintain the “stemness” of embryonic and hematopoietic stem cells. Enhancer of zeste homolog 1 and 2 (EZH1/2) are catalytic components of PRC2, which trimethylate histone H3 at lysine 27 (H3K27me3) to repress transcription of target genes. Mutation and overexpression of EZH2 are associated with many cancers, including MM. Here, we found that expression of EZH1/2 was significantly higher in SP cells than in non-SP cells. These results suggest that overexpression of EZH1/2 is important for maintaining the stemness of MM cells and that both EZH1 and EZH2 are potential therapeutic targets.We developed a novel EZH1/2 dual inhibitor, OR-S1, which potently inhibited both EZH1 and EZH2, and used it to investigate the effect of pharmacologic inhibition of EZH1/2 on MM. OR-S1 suppressed the proliferation of all MM cell lines tested, and its GI50 value was significantly lower than that of the EZH2-specific inhibitor GSK126. Furthermore, flow cytometric analysis revealed that treatment with OR-S1 significantly depleted the SP cells. These results suggest that dual inhibition of EZH1/2 using OR-S1 eradicates myeloma stem cells.RNA sequencing analysis revealed that the transcriptional profiles of OR-S1-treated MM cell lines were characterized by up-regulation of genes related to the Wnt pathway. RT-PCR confirmed that OR-S1 treatment markedly increased expression of Wnt and Frizzled family members. Furthermore, western blot analysis revealed that non-phosphorylated (active) β-catenin was strongly expressed in OR-S1-treated cells. To determine whether there is direct crosstalk between PRC2 and Wnt signaling, we performed chromatin immunoprecipitation (ChIP)-sequencing using MM cell lines. This revealed that most Wnt and Frizzled loci were marked by H3K27me3. Furthermore, ChIP-qPCR analysis demonstrated that dual inhibition of EZH1/2 using OR-S1 markedly decreased the level of H3K27me3 at most of these loci. These results demonstrate that PRC2 directly targets Wnt signaling and that dual inhibition of EZH1/2 using OR-S1 activates canonical Wnt signaling.Increased activation of the canonical Wnt signaling pathway reduces self-renewal and differentiation of hematopoietic stem cells. Therefore, we generated β-catenin-overexpressing MM cells to examine the effects of increased Wnt signaling. Surprisingly, proliferation of these cells was significantly lower than that of control cells. Furthermore, flow cytometric analysis revealed that overexpression of β-catenin significantly depleted the SP cells. These results suggest that over-activation of Wnt signaling induced by dual inhibition of EZH1/2 reduces the proliferation of MM cells by inhibiting self-renewal, which is important to maintain the stemness of MM cells.Oral administration of OR-S1 to mice bearing MM xenografts significantly impaired subcutaneous tumors. Long-term administration of OR-S1 at lower doses to mice bearing orthotopic xenografts eradicated minimal residual disease from bone marrow and completely cured MM, without eliciting any serious side effects (Figure). Furthermore, we developed an orthotopic patient-derived xenograft model using specimens from a relapsed and heavily pretreated MM patient. In this model, OR-S1 treatment reduced the serum levels of human immunoglobulins. These results indicate that EZH1/2-targeting therapy with OR-S1 is also effective against a human MM xenograft.Taken together, our results strongly suggest that dual inhibition of EZH1/2 is a promising therapeutic approach to eradicate myeloma stem cells and could lead to important advances in the treatment of MM. [Display omitted] DisclosuresNakagawa:Daiichi Sankyo Co.: Research Funding. Fujita:Daiichi Sankyo Co.: Research Funding. Honma:Daiichi Sankyo Co., Ltd.: Employment. Araki:Daiichi Sankyo: Employment. Kitabayashi:Daiichi Sankyo Co.: Research Funding.

Additional Inhibition of Wnt/β-Catenin Signaling by Metformin in DAA Treatments as a Novel Therapeutic Strategy for HCV-Infected Patients.

Chronic hepatitis C virus (HCV) infection causes hepatocellular carcinoma (HCC). Although HCV clearance has been improved by the advent of direct-acting antiviral agents (DAA), retrospective studies have shown that the risk of subsequent HCC, while considerably decreased compared with active HCV infection, persists after DAA regimens. However, either the mechanisms of how chronic HCV infection causes HCC or the factors responsible for HCC development after viral eradication in patients with DAA treatments remain elusive. We reported an in vitro model of chronic HCV infection and determined Wnt/β-catenin signaling activation due to the inhibition of GSK-3β activity via serine 9 phosphorylation (p-ser9-GSK-3β) leading to stable non-phosphorylated β-catenin. Immunohistochemical staining demonstrated the upregulation of both β-catenin and p-Ser9-GSK-3β in HCV-induced HCC tissues. Chronic HCV infection increased proliferation and colony-forming ability, but knockdown of β-catenin decreased proliferation and increased apoptosis. Unexpectedly, Wnt/β-catenin signaling remained activated in chronic HCV-infected cells after HCV eradication by DAA, but metformin reversed it through PKA/GSK-3β-mediated β-catenin degradation, inhibited colony-forming ability and proliferation, and increased apoptosis, suggesting that DAA therapy in combination with metformin may be a novel therapy to treat HCV-associated HCC where metformin suppresses Wnt/β-catenin signaling for HCV-infected patients.

Estrogen Receptor Signaling Pathways Involved in Invasion and Colony Formation of Androgen-Independent Prostate Cancer Cells PC-3

Castration-resistant prostate cancer (CRPC) is an advanced and androgen-independent form of prostate cancer. Recent studies of rapid actions mediated by estrogen in the prostate and its relationship with CRPC are emerging. We have previously shown that estrogen receptor (ER) promotes migration and invasion of the androgen-independent prostate cancer cells PC-3, but the signaling pathways involved in these events remain to be elucidated. Therefore, this study aimed to analyze the role of ERα and ERβ in the activation of SRC, and the involvement of SRC and PI3K/AKT on invasion and colony formation of the PC-3 cells. Our results showed that the activation of ERα (using ERα-selective agonist PPT) and ERβ (using ERβ-selective agonist DPN) increased phosphorylation of SRC in PC-3 cells. In the presence of the selective inhibitor for SRC-family kinases PP2, the effects of DPN and PPT on transmigration and soft agar colony formation assays were decreased. Furthermore, SRC is involved in the expression of the non-phosphorylated β-catenin. Finally, using PI3K specific inhibitor Wortmannin and AKT inhibitor MK2206, we showed that PI3K/AKT are also required for invasion and colony formation of PC-3 cells simulated by ER. This study provides novel insights into molecular mechanisms of ER in PC-3 cells by demonstrating that ER, located outside the cell nucleus, activates rapid responses molecules, including SRC and PI3K/AKT, which enhance the tumorigenic potential of prostate cancer cells, increasing cell proliferation, migration, invasion, and tumor formation.

Celastrol attenuates arterial and valvular calcification via inhibiting BMP2/Smad1/5 signalling.

Vascular calcification is an important risk factor for the mortality and morbidity in chronic kidney disease (CKD). Unfortunately, until now there is no certain medication targeting vascular calcification in CKD. In this study, we explored the inhibitory effect of celastrol on high calcium–induced vascular calcification and the underlying molecular mechanisms. Cell proliferation assay showed that celastrol inhibited aortic valve interstitial cell (VIC) and vascular smooth muscle cell (VSMC) proliferation when its concentration was higher than 0.6 μmol/L. 0.8 μmol/L celastrol inhibited the expression of osteogenic genes and calcium deposition induced by high‐calcium medium in both AVICs and VSMCs. In mouse vascular calcification model induced by adenine combined with vitamin D, alizarin red and immunostaining showed that celastrol inhibited pro‐calcification gene expression and calcium deposition in aortic wall and aortic valve tissues. At the molecular level, celastrol inhibited the increase of BMP2, phosphorylated Smad1/5 (p‐Smad1/5) and non‐phosphorylated β‐catenin (n‐p‐β‐catenin) induced by high‐calcium medium both in vitro and in vivo. Also, BMP2 overexpression reversed the anti‐calcification effects of celastrol by recovering the decrease of p‐Smad1/5 and n‐p‐β‐catenin. Furthermore, celastrol prevented the up‐regulation of BMPRII and down‐regulation of Smad6 induced by high calcium, and this protectory effect can be abolished by BMP2 overexpression. In conclusion, our data for the first time demonstrate that celastrol attenuates high calcium–induced arterial and valvular calcification by inhibiting BMP2/Smad1/5 signalling, which may provide a novel therapeutic strategy for arterial and valvular calcification in patients with CKD.

TP53INP2 Modulates Epithelial-to-Mesenchymal Transition via the GSK-3β/β-Catenin/Snail1 Pathway in Bladder Cancer Cells.

BackgroundThe tumor protein p53-inducible nuclear protein 2 (TP53INP2), an autophagy protein, is essential for autophagosome formation. The deregulation of autophagy is associated with multiple human diseases, including cancer. The present study aims to explore the role of TP53INP2 in bladder cancer.Materials and MethodsQuantitative real-time polymerase chain reaction was used to detect the mRNA level. Relative TP53INP2 protein expression was detected by immunohistochemistry and Western blot. The effect of TP53INP2 silencing on the proliferation, migration, and invasion of bladder cancer cells was investigated by CCK-8 detection kit and transwell assay. In addition, transfection and immunofluorescence were performed.ResultsIn this study, we report that high expression of TP53INP2 is correlated with poor patient survival in bladder cancer. Results demonstrate that the depletion of TP53INP2 inhibits the migration, invasion, and epithelial-to-mesenchymal transition (EMT) of bladder cancer cells. The underlying mechanism was explored. Results show that the TP53INP2 knockdown suppresses EMT by inhibiting the active non-phosphorylated β-catenin and decreasing the Snail1 levels. Furthermore, the glycogen synthase kinase-3 beta (GSK-3β) inhibitor IM-12 abrogates the effect of TP53INP2 silencing. Interestingly, the induction of autophagy partially abrogates the TP53INP2 knockdown-induced decrease in active β-catenin and inhibition of migration and invasion in bladder cancer cells.ConclusionIn summary, our results show that the downregulation of TP53INP2 inhibits EMT via the GSK-3β/β-catenin/Snail1 pathway in bladder cancer. The findings of this study uncover the novel role of TP53INP2 and offer new insights into bladder cancer clinical therapy.

R-Spondin 1 (RSPO1) Increases Mouse Intestinal Organoid Unit Size and Survival in vitro and Improves Tissue-Engineered Small Intestine Formation in vivo.

Introduction: Cell therapy and tissue engineering has recently emerged as a new option for short bowel syndrome (SBS) treatment, generating tissue engineered small intestine (TESI) from organoid units (OU) and biodegradable scaffolds. The recombinant human R-Spondin 1 (rhRSPO1) protein may be a key player in this process due to its mitogenic activity in intestinal stem cells.Objective: Aiming at optimizing the TESI formation process and advancing this technology closer to the clinic, we evaluated the effects of rhRSPO1 protein on OU culture and TESI formation.Methods: Intestinal OU were isolated from C57BL/6 mice and cultured in Matrigel in the presence or absence of recombinant human rhRSPO1. Throughout the culture, OU growth and survival rates were evaluated, and cells were harvested on day 3. OU were seeded onto biodegradable scaffolds, in the presence or absence of 5 μg of rhRSPO1 and implanted into the omentum of NOD/SCID mice in order to generate TESI. The explants were harvested after 30 days, weighed, fixed in formalin and embedded in paraffin for histological analysis and immunofluorescence for different cell markers.Results: After 3 days, rhRSPO1-treated OU attained a larger size, when compared to the control group, becoming 5.7 times larger on day 6. Increased survival was observed from the second day in culture, with a 2-fold increase in OU survival between days 3 and 6. A 4.8-fold increase of non-phosphorylated β-catenin and increased relative expression of Lgr5 mRNA in the rhRSPO1-treated group confirms activation of the canonical Wnt pathway and suggests maintenance of the OU stem cell niche and associated stemness. After 30 days of in vivo maturation, rhRSPO1-treated TESI presented a larger mass than constructs treated with saline, developing a more mature intestinal epithelium with well-formed villi and crypts. In addition, the efficiency of OU-loaded rhRSPO1-treated scaffolds significantly increased, forming TESI in 100% of the samples (N = 8), of which 40% presented maximum degree of development, as compared to 66.6% in the control group (N = 9).Conclusion: rhRSPO1 treatment improves the culture of mouse intestinal OU, increasing its size and survival in vitro, and TESI formation in vivo, increasing its mass, degree of development and engraftment.

Inhibition of Wnt/β-Catenin Signaling in Neuroendocrine Tumors in vitro: Antitumoral Effects.

Background and aims: Inhibition of Wnt/β-catenin signaling by specific inhibitors is currently being investigated as an antitumoral strategy for various cancers. The role of Wnt/β-catenin signaling in neuroendocrine tumors still needs to be further investigated. Methods: This study investigated the antitumor activity of the porcupine (PORCN) inhibitor WNT974 and the β-catenin inhibitor PRI-724 in human neuroendocrine tumor (NET) cell lines BON1, QGP-1, and NCI-H727 in vitro. NET cells were treated with WNT974, PRI-724, or small interfering ribonucleic acids against β-catenin, and subsequent analyses included cell viability assays, flow cytometric cell cycle analysis, caspase3/7 assays and Western blot analysis. Results: Treatment of NET cells with WNT974 significantly reduced NET cell viability in a dose- and time-dependent manner by inducing NET cell cycle arrest at the G1 and G2/M phases without inducing apoptosis. WNT974 primarily blocked Wnt/β-catenin signaling by the dose- and time-dependent downregulation of low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation and non-phosphorylated β-catenin and total β-catenin, as well as the genes targeting the latter (c-Myc and cyclinD1). Furthermore, the WNT974-induced reduction of NET cell viability occurred through the inhibition of GSK-3-dependent or independent signaling (including pAKT/mTOR, pEGFR and pIGFR signaling). Similarly, treatment of NET cells with the β-catenin inhibitor PRI-724 caused significant growth inhibition, while the knockdown of β-catenin expression by siRNA reduced NET tumor cell viability of BON1 cells but not of NCI-H727 cells. Conclusions: The PORCN inhibitor WNT974 possesses antitumor properties in NET cell lines by inhibiting Wnt and related signaling. In addition, the β-catenin inhibitor PRI-724 possesses antitumor properties in NET cell lines. Future studies are needed to determine the role of Wnt/β-catenin signaling in NET as a potential therapeutic target.

Bone morphogenetic protein 2 induces the activation of WNT/β-catenin signaling and human trophoblast invasion through up-regulating BAMBI

Both bone morphogenetic protein 2 (BMP2) and WNT/β-catenin signaling promote human trophoblast cell invasion. BMP2 has been shown to up-regulate bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) in granulosa cells. Besides, studies indicate BAMBI is a positive regulator for WNT/β-catenin signaling. However, whether BMP2 can increase BAMBI expression to induce WNT/β-catenin signaling for trophoblast cell invasion is still unknown. To study the roles of BAMBI in BMP2-induced activation of WNT/β-catenin signaling and human trophoblast invasion, we used immortalized human extravillous trophoblast (EVT) cell line (HTR8/SVneo) and primary human EVT cells as study models. Messenger RNA and protein levels of target genes were checked with RT-qPCR and Western blot assay respectively. The function of target proteins was studied via small interfering RNA (siRNA)-mediated knockdown. In addition, trophoblast cell invasiveness was examined by matrigel-coated transwell assays. Our results demonstrate that BMP2 treatment increased BAMBI mRNA levels and the activation of WNT/β-catenin signaling including the raised phosphorylation of GSK3β, the up-regulation of active (non-phosphorylated) β-catenin as well as its downstream target molecule cyclin D1, all of which were totally blocked by the activin receptor-like kinases (ALK) 2/3 inhibitor DMH1 or siRNA-mediated knockdown of BAMBI in HTR8/SVneo cells. Consistently, in primary human EVT cells, BMP2 also induced the up-regulation of BAMBI and the activation of WNT/β-catenin signaling indicated by the increased levels of active β-catenin and cyclin D1, which were completely blocked by BAMBI knockdown. In conclusion, BMP2 promotes the activation of canonical WNT/β-catenin signaling and human trophoblast cell invasion by up-regulating BAMBI.